The present invention relates generally to the construction of fusion proteins that enhance the immune response of an antigen and are efficiently expressed and secreted by yeast host cells. More specifically, the invention relates to yeast recombinant expression systems for producing fusion proteins comprising a granulocyte-macrophage colony-stimulating factor (GM-CSF) domain fused to a selected antigen domain.
Technologies for the efficient production of large quantities of antigenic proteins for use as immunogens have been sought for many years. Genes encoding protein antigens and fragments of antigens comprising particular epitopes have been expressed in prokaryotic and eukaryotic cell expression systems with varying degrees of success. To elicit an antibody response in animals, administration of adjuvants, repeated administration of the expressed protein, or both often were needed.
Certain materials have been shown to have adjuvant activity, including for example alum, fragments of bacterial membranes, liposomes, coupling a protein of interest to a larger immunogenic protein, RIBI, and TiterMax.RTM.. Of all of them, alum is the only adjuvant licensed by the Food and Drug Administration for use in humans. Some investigators have attempted to chemically couple adjuvants to antigens. Such coupling involves harsh treatment and often results in destruction of a portion of the antigen and reduced antigenicity.
Some cytokines, e.g., interleukin-4 (IL-4) and GM-CSF, attract and activate antigen-presenting cells for more efficient presentation of antigens to T cells. These cytokines have been co-administered with antigen to increase antigenic activity. Other studies have shown that the host response to tumor challenge can be increased by inoculation of tumor cells genetically engineered to express particular cytokines, including .gamma.-INF, TNF-.alpha., IL-2, IL-4, IL-6, IL-7, or GM-CSF.
Further, Tao and Levy (Nature, 362: 755-758 (1993)) created chimeric tumor idiotype/GM-CSF fusion proteins as vaccines for B-cell lymphoma. They created their fusion proteins by constructing plasmids with a coding sequence of a heavy-chain variable region from a mouse B-cell tumor inserted upstream of a human IgG1 heavy-chain constant region gene and a restriction cite generated next to the last codon of the CH3 exon into which genetic fragments encoding either murine or human GM-CSF were inserted. These heavy chain vectors were then cotransfected with a light chain chimeric constructs into a malignant plasma cell tumor. The proteins made by the transfected cells were tetrameric proteins that were dimeric with respect to GM-CSF.
Research continues toward enhancing the immune response of an antigen. Proteins that have the bioactivity of both cytokines and antigens will provide the advantages of using chemically defined antigenic entities and eliminating the need to separately administer or co-administer cytokines and antigens or inoculate patients with genetically modified living tumor cells.